Antigen presentation to CD4+ T cells involves antigen uptake into endocytic compartments where unfolded proteins meet with newly synthesized MHC class II molecules. Association of class II molecules with the invariant chain is necessary for their transport to antigen processing compartments. The human invariant chain exists in two forms that differ by the length of their cytoplasmic tails. A new radiolabeling technique was developed to follow the intracellular transport of newly synthesized class II molecules. This technique, more sensitive than any previously available, was used to demonstrate that a cohort of newly synthesized MHC class II molecules is transported directly and rapidly to the cell surface from where it is retrieved by an efficient internalization signal in the cytoplasmic tail of the invariant chain. However, only class II molecules associated with the short-tailed invariant chain were detected at the cell surface. Class II molecules associated with the long-tailed invariant chain are transported directly to endosomes from the trans-Golgi. Two forms of an influenza virus cytosolic antigen with different half-lives were engineered to test whether the endogenous pathway for class II-restricted presentation involves the same cytosolic antigen degradation used for class I-restrictred antigen presentation. In contrast to the class I-restricted presentation, class II molecules presented only the long-lived form of antigen. Thus, rapid degradation of the protein in the cytosol precluded its presentation by class II molecules. These data suggest that the turnover of long-lived cytosolic proteins, some of which is mediated by delivery into endosomal/lysosomal compartments, provides a mechanism for immune surveillance by CD4 T cells.